A method of high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, wherein the sample is subjected to a light intensity distribution including an intensity maximum of focused fluorescence excitation light, wherein partial areas of interest of the sample are scanned with the light intensity distribution, wherein the fluorescence light emitted out of the sample is registered and allocated to the respective location of the light intensity distribution, and wherein the subjection of the sample to the light intensity distribution is terminated for the respective location, when a predetermined maximum light amount of the fluorescence light emitted out of the sample has been registered or even prior to that, if within a predetermined period of time a predetermined minimum light amount of the fluorescence light emitted out of the sample has not been registered, is known from R. A. Hoebe et al.: Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging, Nature Biotechnology, Volume 25, Number 2, February 2007, pages 249 to 253. By this known method, the subjection of the sample to the light intensity distribution is cut back as compared to a subjection over equal periods of time in all locations, everywhere where comparatively many fluorescence markers or no fluorescence markers are located in the sample. The light dose to which the sample is subjected for recording an image of the structure marked with the fluorescence markers may thus be considerably reduced. In the known method, the termination of the subjection of the sample to the light intensity distribution occurs due to controlling an acousto-optical modulator in the beam path of the fluorescence excitation light coming from a laser, the acousto-optical modulator being controlled depending on the light signal of a detector used for registering the fluorescence light.
R. A. Hoebe et al. also describe how the effect of the nevertheless occurring bleaching of the fluorescence markers may be corrected. For this purpose, the light dose to which the pixels of the sample have already been subjected is determined and used for correcting the intensity of the fluorescence light registered.
In a figure, Hoebe et al. document the cumulated light doses which have been received by each individual pixel in the focal plane after the entire volume of a sample has been scanned. In the figure, the different light doses are represented in false colors.
A method of high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, which is also designated as RESCue-STED is known from T. Staudt et al.: Far-field optical nanoscopy with reduced number of state transition cycles, Optics Express Vol. 19, No. 6, 14 Mar. 2011, pages 5644 to 5657. Here, the sample is subjected to a light intensity distribution in which an intensity maximum of focused fluorescence excitation light is superimposed with an intensity minimum of focused fluorescence inhibition light enclosed by intensity maxima of focused fluorescence inhibition light forming a donut. Parts of interest of the sample are scanned with the intensity minimum of the focused fluorescence inhibition light, and fluorescence light emitted out of the sample is registered and allocated to the location of the intensity minimum of the focused fluorescence inhibition light in the sample. The subjection of the sample to the light intensity distribution consisting of the focused fluorescence excitation light and the focused fluorescence inhibition light is terminated for the respective location of the intensity minimum of the focused fluorescence inhibition light, when a predetermined maximum light amount of the fluorescence light emitted out of the sample has been registered or when, even prior to that, a predetermined minimum light amount of the fluorescence light emitted out of the sample has not yet been registered within a predetermined period of time. In this way, the number of cycles of excitation and deexcitation which the fluorescence markers in the sample undergo in recording an image of the sample and thus the danger of bleaching the fluorescence markers are reduced.
T. Staudt et al. also describe an alternative way of implementing their RESCue strategy in which an upfront information with regard to the location of fluorescence markers is used to directly approach those coordinates at which a fluorescence inhibition is needed and to omit those locations at which fluorescence inhibition would be out of function. For example, it is proposed to at first record an image of lower resolution to obtain coarse information on the spatial structure and the density of features in the sample. Some features will then be rather isolated, and others will be densely packed. One may then (successively) apply a higher spatial resolution and concentrate on areas in which dense features are to be resolved. At the same time, coordinates may be omitted at which not objects are to be separated and at which, correspondingly, no switching off by fluorescence inhibition is necessary. The upfront information on the density of the fluorescence markers or the features may be obtained from the brightness of the initial image.
In both ways it is made possible to repeatedly image altering samples or to record images of the same sample along neighboring parallel sections.
In both known methods, the concentration of the fluorescence markers at the respective location of the sample is determined from the ratio of the measured light amount and the associated duration of subjecting the sample to the light intensity distribution, as this duration, in contrast to common laser scanning fluorescence microscopy in which the sample is subjected to the same light intensity distribution for a fixed period of time at each location, varies from location to location.
There still is a need of methods of high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, in which the light dose to which a sample is subjected for recording an image of a desired image quality may even be reduced further.